The goal of the proposed research is to elucidate molecular and physical determinants that influence or control the thermodynamics and catalytic efficiency of assimilatory NADH:nitrate reductase (E.C. 1.6.6.1). Comprising four identical subunits, each of which contains FAD, cytochrome b557 and Mo-pterin in a 1:1:1 stoichiometry, this enzyme catalyzes the rate-limiting and regulated step in the process of inorganic nitrogen assimilation and is an excellent model for investigating electron transfer reactions in complex, multi-center proteins containing diverse prosthetic groups. In addition, this enzyme shares a number of common structural and mechanistic features with a variety of other flavoheme- and Mo-containing proteins including cytochrome b5 reductase, cytochrome b5 and sulfite oxidase. The current grant period has focused on defining the thermodynamic and steady-state kinetic parameters of the native enzymes. The proposed research will focus on identifying determinants of redox potential of the various prosthetic groups within the enzyme and the effects of altering these potentials on catalytic function, in addition to identification of functional groups involved in binding and catalysis. The specific aims of the proposed research are to establish the pathway of electron transfer within the enzyme and to determine the rates of the various intra- and intermolecular electron transfer reactions between the different prosthetic groups in active and substituted forms of Chlorella nitrate reductase using a combination of steady-state, rapid kinetic and electrochemical methods. Relative redox potential modulation of the FAD and Mo-pterin centers will be examined as a mechanism of increasing or decreasing catalytic efficiency. Redox properties will also be examined under various environmental conditions which relate to physiological function, such as in the presence of NADH/NAD+ and NO3-/NO2- or cyanide. In addition, amino acid residues crucial to the catalytic activity of the enzyme will be identified by specific chemical modification studies using both the native enzyme and various recombinant proteins corresponding to the individual functional domains. Specific amino acid modification reagents will be used together with protein sequencing studies to identify cysteine, lysine and arginine residues that are essential to the function of the FAD and Mo-pterin centers. These studies will enhance our understanding of important structure-function relationships of nitrate reductase and other related proteins.